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Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures.

Uchea C, Owen SF, Chipman JK - Toxicol Res (Camb) (2015)

Bottom Line: Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques.Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors.In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux.

View Article: PubMed Central - PubMed

Affiliation: University of Birmingham , School of Biosciences , Birmingham , B15 2TT , UK ; AstraZeneca , Alderley Park , Macclesfield , Cheshire , SK10 4TF , UK . Email: Stewart.Owen@AstraZeneca.com.

ABSTRACT

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants.

No MeSH data available.


Representative visualisation of the accumulation of DHFDA (A), H33342 (B), calcein-AM (C) and rhodamine 123 (D) in hepatocytes in spheroid cultures. A–D 1 show representative controls and A–D 2 show representative spheroids following treatment with sodium taurocholate (A), Ko143 (B), probenecid (C) and cyclosporin A (D). The intracellular accumulation of fluorescent probes in spheroids was observed using confocal microscopy. At the maximum concentrations of inhibitors used, a greater accumulation of fluorescence was observed in spheroids, in comparison to untreated cells in each case. Spheroids used from 15 days post isolation. Images from left to right: fluorescence channel; Bright field scan; merged image. All scale bars indicate 100 μm.
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fig2: Representative visualisation of the accumulation of DHFDA (A), H33342 (B), calcein-AM (C) and rhodamine 123 (D) in hepatocytes in spheroid cultures. A–D 1 show representative controls and A–D 2 show representative spheroids following treatment with sodium taurocholate (A), Ko143 (B), probenecid (C) and cyclosporin A (D). The intracellular accumulation of fluorescent probes in spheroids was observed using confocal microscopy. At the maximum concentrations of inhibitors used, a greater accumulation of fluorescence was observed in spheroids, in comparison to untreated cells in each case. Spheroids used from 15 days post isolation. Images from left to right: fluorescence channel; Bright field scan; merged image. All scale bars indicate 100 μm.

Mentions: The functions of the efflux transporters of interest were assessed using fluorescent substrates and inhibitors successfully employed in other studies (e.g.ref. 29, 32, 46, 49 and 55). The fluorescent substrates used were calcein-acetoxymethylester (Ca-AM), rhodamine 123 (Rh123), hoechst 33342 (H33342) and 2′,7′-dichlorodihydrofluorescein diacetate (DHFDA). The intracellular presence and accumulation of these compounds in spheroids was observed and confirmed by confocal microscopy (Fig. 2). Increases in intracellular accumulation of substrates as a result of specific inhibition were quantified using a fluorescence plate reader and compared to the fluorescence measurements recorded in control cells. All inhibitors investigated caused significant changes in substrate accumulation.


Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures.

Uchea C, Owen SF, Chipman JK - Toxicol Res (Camb) (2015)

Representative visualisation of the accumulation of DHFDA (A), H33342 (B), calcein-AM (C) and rhodamine 123 (D) in hepatocytes in spheroid cultures. A–D 1 show representative controls and A–D 2 show representative spheroids following treatment with sodium taurocholate (A), Ko143 (B), probenecid (C) and cyclosporin A (D). The intracellular accumulation of fluorescent probes in spheroids was observed using confocal microscopy. At the maximum concentrations of inhibitors used, a greater accumulation of fluorescence was observed in spheroids, in comparison to untreated cells in each case. Spheroids used from 15 days post isolation. Images from left to right: fluorescence channel; Bright field scan; merged image. All scale bars indicate 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384106&req=5

fig2: Representative visualisation of the accumulation of DHFDA (A), H33342 (B), calcein-AM (C) and rhodamine 123 (D) in hepatocytes in spheroid cultures. A–D 1 show representative controls and A–D 2 show representative spheroids following treatment with sodium taurocholate (A), Ko143 (B), probenecid (C) and cyclosporin A (D). The intracellular accumulation of fluorescent probes in spheroids was observed using confocal microscopy. At the maximum concentrations of inhibitors used, a greater accumulation of fluorescence was observed in spheroids, in comparison to untreated cells in each case. Spheroids used from 15 days post isolation. Images from left to right: fluorescence channel; Bright field scan; merged image. All scale bars indicate 100 μm.
Mentions: The functions of the efflux transporters of interest were assessed using fluorescent substrates and inhibitors successfully employed in other studies (e.g.ref. 29, 32, 46, 49 and 55). The fluorescent substrates used were calcein-acetoxymethylester (Ca-AM), rhodamine 123 (Rh123), hoechst 33342 (H33342) and 2′,7′-dichlorodihydrofluorescein diacetate (DHFDA). The intracellular presence and accumulation of these compounds in spheroids was observed and confirmed by confocal microscopy (Fig. 2). Increases in intracellular accumulation of substrates as a result of specific inhibition were quantified using a fluorescence plate reader and compared to the fluorescence measurements recorded in control cells. All inhibitors investigated caused significant changes in substrate accumulation.

Bottom Line: Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques.Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors.In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux.

View Article: PubMed Central - PubMed

Affiliation: University of Birmingham , School of Biosciences , Birmingham , B15 2TT , UK ; AstraZeneca , Alderley Park , Macclesfield , Cheshire , SK10 4TF , UK . Email: Stewart.Owen@AstraZeneca.com.

ABSTRACT

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants.

No MeSH data available.